Removal of neuroblastoma cells from bone marrow with monoclonal antibodies conjugated to magnetic microspheres.

Monoclonal antibodies bound to polystyrene microspheres containing magnetite have been used to remove tumour cells from bone marrow destined for autologous transplantation. The small magnetic beads can be targeted to the surface of tumour cells to render these magnetic. A flow system using permanent samarium cobalt magnets effects the rapid and efficient removal of "magnetic" tumour cells from bone marrow. The system is designed to be used with any type of tumour cell, so that by changing panels of monoclonal antibody it can be used for "cleaning" the bone marrow in many different malignancies.

Cell labeling and magnetic separation by means of immunoreagents based on polyacrolein microspheres.

Polyacrolein (PA) microspheres were synthesized by means of ionizing radiation and shown to contain aldehyde groups which form covalent bounds with amino compounds and proteins. PA microspheres made fluorescent after reaction with fluorescein-labeled antibodies were found to specifically label sensitized sheep red blood cells (SRBC). PA microspheres could also be grafted onto a variety of polymeric spheres of different sizes and composition by ionizing radiation. These hybrid spheres, i.e., preformed polymeric spheres with PA microspheres grafted on their surfaces could bind antibodies which retained specificity of reaction with cell surface receptors. Purification of sensitized SRBC from a mixture containing chicken red blood cells (CRBC) by means of hybrids magnetic spheres in a magnetic field was demonstrated.

Synthesis of adriamycin-coupled polyglutaraldehyde microspheres and evaluation of their cytostatic activity.

Adriamycin was coupled to polyglutaraldehyde microspheres having an average diameter of 4500 A. The coupled microspheres remained stable during incubation with cells. Full cytostatic activity was observed when the coupled adriamycin was tested with murine or human leukemia and murine sarcoma cell lines. A 10-fold increase in sensitivity was obtained with drug-resistant human leukemia cell lines. Repeated use of the coupled microspheres in the cytostatic assays did not increase their activity, indicating that these complexes can be recycled. The results suggest that coupled adriamycin sufficiently perturbs the plasma membrane to lead to cytostatic activity. It is proposed that this mode of drug delivery provides multiple and repetitious sites for drug-cell interactions. In addition, the drug-polymer complexes may overcome those forms of resistance that are the result of decreased drug binding at the cell surface.

Immunomicrospheres: reagents for cell labeling and separation.

Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicropheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (i) new types of radioimmune or immunofluorescent assays, (ii) improved fluorescence microscopy, (iii) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (iv) markers for use in several types of electron or standard light microscopy, and (v) delivery of lethal compounds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

Electrophoretic cell separation by means of microspheres.

The electrophoretic mobility of fixed human erythrocytes immunologically labeled with poly(vinylpyridine) or poly(glutaraldehyde) microspheres was reduced by approximately 40%. This observation was utilized in preparative scale electrophoretic separations of fixed human and turkey erythrocytes, the mobilities of which under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. We suggest that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distributions, may be significantly enhanced by immunospecific labeling of target populations using microspheres.

Polyglutaraldehyde: a new reagent for coupling proteins to microspheres and for labeling cell-surface receptions. II. Simplified labeling method by means of non-magnetic and magnetic polyglutaraldehyde microspheres.

Procedures were developed for the synthesis of a new immunoreagent in form of polyglutaraldehyde (PGL) microspheres in sizes ranging from about 50 nm to 1.5 micron. Addition of fluorochromes during synthesis yielded microspheres of high fluorescence intensity. By carrying out the polymerization of glutaraldehyde in presence of iron oxide, magnetic PGL microspheres were produced. Antibody conjugates obtained by interaction of PGL microspheres with immunoglobulins were used to label human red blood cells (RBC) and lymphocytes. A simple method for the separation of magnetically labeled human RBC from unlabeled cells was demonstrated.

Polyglutaraldehyde: a new reagent for coupling proteins to microspheres and for labeling cell-surface receptors.

Glutaraldehyde polymerized in basic aqueous solutions was found to react with low molecular weight amines, immunoglobulins and hemoglobin. The polyglutaraldehyde was covalently bound to hypdrophilic microspheres. The rate of addition of proteins to the polyglutaraldehyde-derivatized microspheres was investigated spectrophotometrically as a function of pH and temperature. The reaction of polyglutaraldehyde was found to be faster than that of the monomer. The findings led to successful labeling of human lymphocyte subpopulations.

Human B lymphocytes in giemsa stained preparations.

A mixed antiglobulin procedure has been developed to provide simultaneous study of cell surface immunoglobulin and detailed cytological features in Giemsa stained preparations. The percentage of surface immunoglobulin bearing lymphocytes, monocytes and granulocytes changed dramatically when lymphocytes were incubated at 37 degrees C, or following sheep erythrocyte rosette enrichment procedures.

Interaction of living cells with polyionenes and polyionene-coated surfaces.

Polyionenes have been shown recently (A. Rembaum, Appl. Polym. Symp. No. 22, 299, 1973) to produce the following biological effects: 1) bactericidal action, 2) formation of insoluble complexes with DNA and heparin, 3) neuromuscular blocking action, 4) cell aggregation and lysis, and (5) cell adhesion. In present study, polyionenes of various structures (mainly I3, 3, I6, 10) were used as molecular probes to gain an understanding of the cell surface phenomena of adhesion on glass- and polyionenes-treated surfaces. Since tumor cells show different durface cell properties, including an increase in the anodic mobility, they bind preferentially to polyionene-treated surfaces. Normal human diploid WI-38 cells were found to adhere at a lower rate than SV-transformed WI-38 cells. However, cell spreading was accelerated in both cases. A study of the interaction of polyionenes in solution in vitro and in vivo and polyionenes covalently bound to polymeric microspheres with leukemic murine EL4 cells and normal thymocytes showed specific cytotoxity towards the leukemic cells.

Reactions of human platelets with microspheres of poly(hydroxymethyl methacrylate) and polyacrylamide.

We have studied the contact interaction of platelets with hydrogels. In the form of microspheres, 0.6-1.0 mu, poly(glycol methacrylate) (polyHEMA) and poly(methyl methacrylate) beads cause platelets to aggregate at concentrations of about 10(8) beads/ml. Polyacrylamide and (20/80) poly(acrylamide-HEMA) copolymer were ineffective in aggregating platelets. The admixture of 20% methacrylate to polyHEMA rendered the beads inactive. Blood plasma components other than fibrinogen were found essential to the interaction of the beads with platelets. Near-infrared spectra of the hydrogels polyacrylamide and polyHEMA showed the water hydrogen bonds to be the same for both and different from those in pure water. The monomer HEMA is an inhibitor of platelet aggregation and the release reaction at levels of 0.1%. It is concluded that the two hydrogels have different blood compatibilities, which depend more on the network structures than the water structures in the respective gels.

Functional polymeric microspheres based on 2-hydroxyethyl methacrylate for immunochemical studies.

Co gamma irradiation of 2-hydroxethyl methacrylate in the presence or in the absence of other acrylic monomers was found to constitute an effective technique for the synthesis of hydrophilic functional microspheres in the size range of approxumately 0.3 to 3 mu in diameter. The effect of monomer concentration, steric stabilization, and electrostatic interaction on the particle size was investigated. Experimental conditions were determined to obtain desired particle sizes of relatively narrow distribution. It was shown that particles may be formed without intermediate micelles, i.e., by homogeneous nucleation, and the rate of particle formation is affected primarily by the rate of particle coalescence in the initial stages of the reaction. When covalently bound to antibodies these microspheres were successfully used to label murine and human lymphocytes.